458 STATE BOARD OF AGRICULTURE. 



ADDITIONAL WORK UPON THE ASSOCIATIVE ACTION OF BACTERIA IN 



THE SOURING OF MILK. 



BY CHARLES E. MARSHALL. 



[Special Bulletin No. 29.] 



In the first article of these studies published in the Centralblatt fiir Bakteri- 

 ologie, II. Bd. XI No. "V;; and as Special Bulletin No. 23 from Michigan Stat© 

 Agricultural College Experiment Station, there was demonstrated conclusively by 

 repeated experiments, the influence of a distinct species of micro-organisms, dia- 

 metrically opposed in its nature and functions to lactic acid bacteria, upon the- 

 souring of milk. In this article, as in the previous discussion, the micro-organ- 

 isms will be designated in the same manner — that micro-organism, which pepton- 

 izes the casein, after a time renders the milk quite alkaline, and is employed for 

 association with the lactic acid bacterium, has been and still continues to be 

 designated by B, and the lactic acid bacterium by A. 



Since recording the first work some fifty tests have been independently made- 

 by as many individuals, all of which tends to strengthen the writer's position 

 in this matter. Farther than this, other micro-organisms which may be classed 

 In general with B have been met which are capable of producing similar re- 

 sults when associated with A in milk, and also others which do not hasten 

 the souring of milk or stimulate the activity of the lactic acid bacteria, but 

 on the other hand, retard or check the souring process. Very dissimilar 

 processes are met, each individually characteristic, yet all deviate in one way 

 and another from the simple process of milk souring in a pure milk culture 

 of lactic acid bacteria designated as A. In this connection therefore two- 

 groups present themselves — those which hasten the souring of milk and those 

 which retard. At this stage of our work it cannot be stated which is the pre- 

 dominating group. As far as the investigations go, that which hastens the 

 process is in the ascendency and the amount of influence exerted is widely 

 varying. 



The immediate cause for this stimulating influence upon the lactic acid 

 bacteria through the development of a species of micro-organism, so different 

 in its effect upon milk and in its nature, calls for consideration. The fact that 

 micro-organism B usually dies out within fifty hours when associated with 

 micro-organism A in milk, indicates that the influencing factor must be the 

 outcome of the first hours of development. Micro-organism B predominates at 

 first but is superseded by A, hence micro-organism B probably produces this 

 factor. If this factor or substance be stable and remain unchanged by heat, 

 it follows that the substance would yield to sterilization and could be used in- 

 dependent of the germs producing it. In accordance with this view, micro- 

 organism B was cultivated in sterile litmus milk culture for forty-eight hours 

 at 23° C, when it became apparent that a change had begun in the milk. The 

 cultures were then heated in steam for three consecutive days, at the end of 

 which time bouillon culture tests made from the milk established the fact of 

 sterility. Sterile litmus milk flasks, from the same lot of milk used for the 

 above cultures of micro-organism B, and containing the same amount of milk 

 in each, were inoculated with micro-organism A in identical amounts as were 

 inoculated into the set of sterilized flask cultures in milk as described. These 

 two sets of flask-cultures, one set in which micro-organism B had grown for 

 forty-eight hours then sterilized and inoculated with micro-organism A, the 

 other set simply inoculated with micro-organism A, were placed at 23° C. and 

 observed from time to time. It was found that micro-organism B had in some 

 manner produced a substance or condition which remained unchanged through 

 sterilization and was apparently as active in stimulating the lactic acid bacteria 

 as the living micro-organisms themselves. To further test this product of 



